OP0209 IMPACT OF CHRONIC INFLAMMATION AND COLLAGEN IV FRAGMENT CANSTATIN ON RHEUMATOID ARTHRITIS SYNOVIAL FIBROBLASTS AND ENDOTHELIAL CELL INTERACTIONS IN VITRO AND IN VIVO
Authors: C. Heck, S. Haun, D. Kürsammer, K. Frommer, M. Arnold, S. Rehart, U. Müller-Ladner, E. Neumann
Journal: Annals of the Rheumatic Diseases (2023)

<h3>Background</h3> Increased neovascularization in the inflamed synovium of patients with rheumatoid arthritis (RA) is a pathological feature in the pathogenesis of RA. Key players in RA progression are chronically activated RA synovial fibroblasts (RASF), which secrete proinflammatory factors (e.g. IL-6), proangiogenic VEGF, IL-11 and matrix metalloproteases (MMP) promoting inflammation, synovial angiogenesis, and invasion of cartilage and bone by degrading the consecutive matrix, resulting in the release of the anti-angiogenic collagen IV fragment canstatin. Canstatin may further activate RASF leading to altered expression of vascular markers such as angiopoietin-2 (ANGPT2) and neovascularization. <h3>Objectives</h3> Analysis of repetitively stimulated RASF- and canstatin-mediated effects on vessel formation in the tube formation assay, in the SCID-mouse model for RA and in synovial tissue of RA patients with respect to ANGPT2 expression and RASF-endothelial cell (EC) interactions. <h3>Methods</h3> 2D tube formation assay was performed using HUVEC seeded on Matrigel. 15% Calcein AM-stained RASF were added. RASF/HUVEC were treated with 0.5µg/ml canstatin. HUVEC were pre-treated with 0.2µg/ml canstatin. RASF were repetitively stimulated three times with 0.05ng/µl IL-1ß every 24h starting at day 2. Tube thickness and the area covered by the formed cellular network were measured. RNA was extracted from tubes and ANGPT2 expression was analysed by qPCR. IL-6 and IL-11 in supernatants were measured by ELISA. Healthy cartilage was subcutaneously co-implanted with RASF into SCID mice. Contralaterally, healthy cartilage without RASF was implanted. Vessel formation was evaluated after 3-45 days. RA and osteoarthritis (OA) synovial tissue was stained for ANGPT2 and CD31. <h3>Results</h3> RASF or HUVEC stimulated once showed a significant IL-6 increase compared to unstimulated controls. After subsequent repetitive stimulation of RASF or HUVEC, a significant decrease in IL-6 (1st stimulation: 5076±1730pg/ml vs. 3rd: 1890±758pg/ml, p&lt;0.0001/ 1st: 488±186pg/ml vs. 3rd: 382±109pg/ml, p=0.041) and IL-11 in RASF (1st: 451±205pg/ml vs. 3rd: 69±51pg/ml, p&lt;0.0001) compared to the first stimulation was observed. Repetitive stimulation of HUVEC+RASF resulted in a significant IL-6 increase for each subsequent stimulation (1st vs. 3rd: p=0.02). RASF significantly reduced tube thickness (22.9µm (SD=6.3) vs. 16.6µm (SD=2.2), p=0.014) and the network area (p&lt;0.0001) and significantly increased ANGPT2 2-fold compared to HUVEC alone. RASF stimulated only once further reduced the network area (p=0.038), while repetitive stimulation significantly attenuated the proinflammatory effect (IL-6, p=0.029). Stimulation of pre-treated HUVEC and unstimulated RASF with canstatin led to disturbed tube formation with reduced tube thickness from 22.9 to 16.9µm (SD=4.4, p=0.011). Co-culture of RASF with pre-treated HUVEC with canstatin further increased the RASF-mediated effect by reducing tube thickness (22.9 to 14.6µm (SD=1.4), p&lt;0.001), with a significant 1.6-fold decrease of ANGPT2. In human RA synovium, ANGPT2 was also found to be significantly upregulated in vessels compared to OA tissue. In SCID mice, RASF-mediated altered vessel formation started at day 3. Helix-like vessels were detectable at early time points of vessel formation in implants with RASF (d3-9), e.g. at day 3, 60% of vessels were helix-like. <h3>Conclusion</h3> RASF induced helix-like vessels in the SCID mouse model suggest an influence on vessel formation. This could be confirmed in vitro as well as on molecular level inducing the vascular marker ANGPT2 in newly formed tubes. ANGPT2-upregulation in vessels was also observed in human RA synovium. Repetitive stimulation of RASF resulted in an inflammatory adjustment to IL-1ß, leading to a significant reduction of IL-6 and improved neovascularization in vitro. The anti-angiogenic canstatin could in part enhance the RASF-mediated effect and was not able to restore RASF-altered tube formation in vitro. <h3>REFERENCES:</h3> NIL. <h3>Acknowledgements:</h3> NIL. <h3>Disclosure of Interests</h3> None Declared.